A longitudinal cohort study of protease inhibitor-naive, HIV-infected individuals was designed to identify genetic variables in viral Gag and envelope sequences associated with response to antiretroviral therapy.

Methods

Genetic and statistical models, including amino acid profiles, phylogenetic analyses, receiver operating characteristic analyses, and covariation analyses, were used to evaluate viral sequences and clinical variables from individuals who developed immune reconstitution with or without suppression of viral replication.

Human immunodeficiency virus intra-host recombination has never been studied in vivo both during early infection and throughout disease progression. The CD8-depleted rhesus macaque model of neuroAIDS was used to investigate the impact of recombination from early infection up to the onset of neuropathology in animals inoculated with a simian immunodeficiency virus (SIV) swarm. Several lymphoid and non-lymphoid tissues were collected longitudinally at 21 days post-infection (p.i.), 61 days p.i. and necropsy (75–118 days p.i.) from four macaques that developed SIV-encephalitis or meningitis, as well as from two animals euthanized at 21 days p.i. The number of recombinant sequences and breakpoints in different tissues and over time from each primate were compared. Breakpoint locations were mapped onto predicted RNA and protein secondary structures. Recombinants were found at each time point and in each primate as early as 21 days p.i. No association was found between recombination rates and specific tissue of origin. Several identical breakpoints were identified in sequences derived from different tissues in the same primate and among different primates. Breakpoints predominantly mapped to unpaired nucleotides or pseudoknots in RNA secondary structures, and proximal to glycosylation sites and cysteine residues in protein sequences, suggesting selective advantage in the emergence of specific recombinant sequences. Results indicate that recombinant sequences can become fixed very early after infection with a heterogeneous viral swarm. Features of RNA and protein secondary structure appear to play a role in driving the production of recombinants and their selection in the rapid disease model of neuroAIDS.

The origin and evolution of HIV-1 in breast milk is unclear, despite the continuing significance of this tissue as a transmitting compartment. To elucidate the evolutionary trajectory of viral populations in a transient mucosal compartment, longitudinal sequences of the envelope gp120 region from plasma and breast milk spanning the first year after delivery were analyzed in six women infected by HIV-1 subtype C.

Methods

Multiple phylogenetic algorithms were used to elucidate the evolutionary history and spatial structure of virus populations between tissues.

Results

Overall persistent mixing of viral sequences between plasma and breast milk indicated that breast milk is not a distinct genetic viral compartment. Unexpectedly, longitudinal phylogenies showed multiple lineages defined by long branches that included virus from both the breast milk and the plasma. Plasma was unlikely the anatomical origin of the most recent common ancestor (MRCA) in at least three of the subjects, while in other women, the temporal origin of the MRCA of the viral populations following delivery occurred well before the onset of breast milk production.

Conclusions

These findings suggest that during pregnancy/lactation, a viral variant distinct from the plasma virus initially seeds the breast milk, followed by subsequent gene flow between the plasma and breast milk tissues. This study indicates the potential for reactivation or re-introduction of distinct lineages during major immunological disruptions during the course of natural infection.

Cross-sectional and longitudinal cellular and soluble plasma markers for inflammation were evaluated in 14 healthy and 33 perinatally HIV-1-infected pediatric subjects prior to and over 96 weeks of protease-inhibitor-containing combination antiretroviral treatment [ART]. All HIV-1-infected subjects reconstituted CD4 T cells either with suppression of viremia or rebound of drug-resistant virus.

Microbial translocation was detected in healthy infants but resolved with age (P<0.05). LPS and sCD14 levels were elevated in all HIV-1 infected subjects (P<0.05 and P<0.0001, respectively) and persisted even if CD4 T cells were fully reconstituted, virus optimally suppressed, and lymphocyte activation resolved by ART. Children with CD4 T cell reconstitution but viral rebound following ART continued to display high levels of sCD27.

Substance use by youth living with HIV (YLWH) is a concern, given potential interactions with virus-associated immune suppression and adverse effects on risk behaviors, neurocognition, and adherence. Self-report substance use measures provide efficient cost-effective assessments. Analyses describe self-reported substance use among YLWH and examine agreement with toxicology assays.

Methods

Seventy-eight youth age 18–24 years (87% male, 71% African–American) with behaviorally acquired HIV-1 infection and 55 uninfected youth completed the Alcohol, Smoking, and Substance Involvement Screening Test to assess drug use frequency, including tobacco, marijuana, cocaine, and alcohol, over the prior three months. Elisa-based toxicology assays were used to detect 27 substances in plasma. Chi-square tests compared substance use between YLWH and uninfected youth; Kappa statistics compared agreement between self-report and toxicology.

Results

YLWH reported marijuana (49%), tobacco (56%), and alcohol (87%) use, with 20%, 28% and 3% reporting daily use of each substance, respectively; other substance use was uncommon. Uninfected youth reported less tobacco use but otherwise similar substance use. All youth who reported daily use of marijuana or tobacco had positive plasma toxicology results, while concordance decreased with less frequent self-reported use. Among youth reporting no substance use, few tested positive (4% YLWH, 2% uninfected youth for cannabis; 8%YLWH for tobacco).

Conclusions

Youth report high rates of marijuana, tobacco, and alcohol use. Concordance between self-report and toxicology for marijuana and tobacco use, particularly for daily users, supports self-report as a valid indicator of substance use in research studies of youth with or without HIV-1 infection.

Next generation sequencing (NGS) is superseding Sanger technology for analysing intra-host viral populations, in terms of genome length and resolution. We introduce two new empirical validation data sets and test the available viral population assembly software. Two intra-host viral population ‘quasispecies’ samples (type-1 human immunodeficiency and hepatitis C virus) were Sanger-sequenced, and plasmid clone mixtures at controlled proportions were shotgun-sequenced using Roche's 454 sequencing platform. The performance of different assemblers was compared in terms of phylogenetic clustering and recombination with the Sanger clones. Phylogenetic clustering showed that all assemblers captured a proportion of the most divergent lineages, but none were able to provide a high precision/recall tradeoff. Estimated variant frequencies mildly correlated with the original. Given the limitations of currently available algorithms identified by our empirical validation, the development and exploitation of additional data sets is needed, in order to establish an efficient framework for viral population reconstruction using NGS.

Extended immune phenotyping of CD4+ and CD8+ T cells subsets were combined with TREC levels and measures of TCR repertoire perturbations in CD8+ T cell subpopulation in order to define the global effect of HIV-1 on T cell dynamics. Evaluations before, and after therapy, were performed in HIV-infected children and compared to healthy individuals.

Regions of HIV-1 gag between p2 and p6Gag/p6Pol, in addition to protease (PR), develop genetic diversity in HIV-1 infected individuals who fail to suppress virus replication by combination protease inhibitor (PI) therapy. To elucidate functional consequences for viral replication and PI susceptibility by changes in Gag that evolve in vivo during PI therapy, a panel of recombinant viruses was constructed. Residues in Gag p2/p7NC cleavage site and p7NC, combined with residues in the flap of PR, defined novel fitness determinants that restored replicative capacity to the posttherapy virus. Multiple determinants in Gag have a dominant effect on PR phenotype and increase susceptibility to inhibitors of drug-resistant or drug-sensitive PR genes. Gag determinants of drug sensitivity and replication alter the fitness landscape of the virus, and viral replicative capacity can be independent of drug sensitivity. The functional linkage between Gag and PR provides targets for novel therapeutics to inhibit drug-resistant viruses.

Infection of CD8-depleted rhesus macaques with the genetically heterogeneous simian immunodeficiency virus (SIV)mac251 viral swarm provides a rapid-disease model for simian acquired immune deficiency syndrome and SIV-encephalitis (SIVE). The objective was to evaluate how the diversity of the swarm influences the initial seeding of the infection that may potentially affect disease progression. Plasma, lymphoid and non-lymphoid (brain and lung) tissues were collected from two infected macaques euthanized at 21 days post-infection (p.i.), as well as longitudinal specimens and post-mortem tissues from four macaques followed throughout the infection. About 1300 gp120 viral sequences were obtained from the infecting SIVmac251 swarm and the macaques longitudinal and post-mortem samples. Phylogenetic and amino acid signature pattern analyses were carried out to assess frequency, transmission dynamics and persistence of specific viral clusters. Although no significant reduction in viral heterogeneity was found early in infection (21 days p.i.), transmission and replication of SIV variants was not entirely random. In particular, two distinct motifs under-represented (<4 %) in the infecting swarm were found at high frequencies (up to 14 %) in all six macaques as early as 21 days p.i. Moreover, a macrophage tropic variant not detected in the viral swarm (<0.3 %) was present at high frequency (29–100 %) in sequences derived from the brain of two macaques with meningitis or severe SIVE. This study demonstrates the highly efficient transmission and persistence in vivo of multiple low frequency SIVmac251 founder variants, characterized by specific gp120 motifs that may be linked to pathogenesis in the rapid-disease model of neuroAIDS.

Chronic hepatitis C virus (HCV) infection can lead to liver cirrhosis in up to 20% of individuals, often requiring liver transplantation. Although the new liver is known to be rapidly reinfected, the dynamics and source of the reinfecting virus(es) are unclear, resulting in some confusion concerning the relationship between clinical outcome and viral characteristics. To clarify the dynamics of liver reinfection, longitudinal serum viral samples from 10 transplant patients were studied. Part of the E1/E2 region was sequenced, and advanced phylogenetic analysis methods were used in a multiparameter analysis to determine the history and ancestry of reinfecting lineages. Our results demonstrated the complexity of HCV evolutionary dynamics after liver transplantation, in which a large diverse population of viruses is transmitted and maintained for months to years. As many as 30 independent lineages in a single patient were found to reinfect the new liver. Several later posttransplant lineages were more closely related to older pretransplant viruses than to viruses detected immediately after transplantation. Although our data are consistent with a number of interpretations, the persistence of high viral genetic variation over long periods of time requires an active mechanism. We discuss possible scenarios, including frequency-dependent selection or variation in selective pressure among viral subpopulations, i.e., the population structure. The latter hypothesis, if correct, could have relevance to the success of newer direct-acting antiviral therapies.

Deep sequencing provides the basis for analysis of biodiversity of taxonomically similar organisms in an environment. While extensively applied to microbiome studies, population genetics studies of viruses are limited. To define the scope of HIV-1 population biodiversity within infected individuals, a suite of phylogenetic and population genetic algorithms was applied to HIV-1 envelope hypervariable domain 3 (Env V3) within peripheral blood mononuclear cells from a group of perinatally HIV-1 subtype B infected, therapy-naïve children.

Results

Biodiversity of HIV-1 Env V3 quasispecies ranged from about 70 to 270 unique sequence clusters across individuals. Viral population structure was organized into a limited number of clusters that included the dominant variants combined with multiple clusters of low frequency variants. Next generation viral quasispecies evolved from low frequency variants at earlier time points through multiple non-synonymous changes in lineages within the evolutionary landscape. Minor V3 variants detected as long as four years after infection co-localized in phylogenetic reconstructions with early transmitting viruses or with subsequent plasma virus circulating two years later.

Conclusions

Deep sequencing defines HIV-1 population complexity and structure, reveals the ebb and flow of dominant and rare viral variants in the host ecosystem, and identifies an evolutionary record of low-frequency cell-associated viral V3 variants that persist for years. Bioinformatics pipeline developed for HIV-1 can be applied for biodiversity studies of virome populations in human, animal, or plant ecosystems.

Infecting rhesus macaques (Macaca mulatta) with the simian immunodeficiency virus (SIV) is an established animal model of human immunodeficiency virus (HIV) pathogenesis. Many studies have used various derivatives of the SIVmac251 viral swarm to investigate several aspects of the disease, including transmission, progression, response to vaccination, and SIV/HIV-associated neurological disorders. However, the lack of standardization of the infecting inoculum complicates comparative analyses. We investigated the genetic diversity and phylogenetic relationships of the 1991 animal-titered SIVmac251 swarm, the peripheral blood mononuclear cell (PBMC) passaged SIVmac251, and additional SIVmac251 sequences derived over the past 20 years. Significant sequence divergence and diversity were evident among the different viral sources. This finding highlights the importance of characterizing the exact source and genetic makeup of the infecting inoculum to achieve controlled experimental conditions and enable meaningful comparisons across studies.

HIV-1 CRF02_AG accounts for >50% of infected individuals in Cameroon. CRF02_AG prevalence has been increasing both in Africa and Europe, particularly in Italy because of migrations from the sub-Saharan region. This study investigated the molecular epidemiology of CRF02_AG in Cameroon by employing Bayesian phylodynamics and analyzed the relationship between HIV-1 CRF02_AG isolates circulating in Italy and those prevalent in Africa to understand the link between the two epidemics. Among 291 Cameroonian reverse transcriptase sequences analyzed, about 70% clustered within three distinct clades, two of which shared a most recent common ancestor, all related to sequences from Western Africa. The major Cameroonian clades emerged during the mid-1970s and slowly spread during the next 30 years. Little or no geographic structure was detected within these clades. One of the major driving forces of the epidemic was likely the high accessibility between locations in Southern Cameroon contributing to the mobility of the population. The remaining Cameroonian sequences and the new strains isolated from Italian patients were interspersed mainly within West and Central African sequences in the tree, indicating a continuous exchange of CRF02_AG viral strains between Cameroon and other African countries, as well as multiple independent introductions in the Italian population. The evaluation of the spread of CRF02_AG may provide significant insight about the future dynamics of the Italian and European epidemic.

Next Generation sequencing (NGS) applied to human papilloma viruses (HPV) can provide sensitive methods to investigate the
molecular epidemiology of multiple type HPV infection. Currently a genotyping system with a comprehensive collection of
updated HPV reference sequences and a capacity to handle NGS data sets is lacking. HPV-QUEST was developed as an automated
and rapid HPV genotyping system. The web-based HPV-QUEST subtyping algorithm was developed using HTML, PHP, Perl
scripting language, and MYSQL as the database backend. HPV-QUEST includes a database of annotated HPV reference sequences
with updated nomenclature covering 5 genuses, 14 species and 150 mucosal and cutaneous types to genotype blasted query
sequences. HPV-QUEST processes up to 10 megabases of sequences within 1 to 2 minutes. Results are reported in html, text and
excel formats and display e-value, blast score, and local and coverage identities; provide genus, species, type, infection site and risk
for the best matched reference HPV sequence; and produce results ready for additional analyses.

Since recombination leads to the generation of mosaic genomes that violate the assumption of traditional phylogenetic methods that sequence evolution can be accurately described by a single tree, results and conclusions based on phylogenetic analysis of data sets including recombinant sequences can be severely misleading. Many methods are able to adequately detect recombination between diverse sequences, for example between different HIV-1 subtypes. More problematic is the identification of recombinants among closely related sequences such as a viral population within a host. We describe a simple algorithmic procedure that enables detection of intra-host recombinants based on split-decomposition networks and a robust statistical test for recombination. By applying this algorithm to several published HIV-1 datasets we conclude that intra-host recombination was significantly underestimated in previous studies and that up to one-third of the env sequences longitudinally sampled from a given subject can be of recombinant origin. The results show that our procedure can be a valuable exploratory tool for detection of recombinant sequences before phylogenetic analysis, and also suggest that HIV-1 recombination in vivo is far more frequent and significant than previously thought.

We sought to investigate the evolutionary and historical reasons for the different epidemiological patterns of HIV-1 in the early epidemic. In order to characterize the demographic history of HIV-1 subtypes A and D in east Africa, we examined molecular epidemiology, geographical and historical data.

Methodology

We employed high-resolution phylodynamics to investigate the introduction of HIV-1A and D into east Africa, the geographic trends of viral spread, and the demographic growth of each subtype. We also used geographic information system data to investigate human migration trends, population growth, and human mobility.

Results

HIV-1A and D were introduced into east Africa after 1950 and spread exponentially during the 1970s, concurrent with eastward expansion. Spatiotemporal data failed to explain the establishment and spread of HIV based on urban population growth and migration. The low prevalence of the virus in the Democratic Republic of Congo before and after the emergence of the pandemic was, however, consistent with regional accessibility data, highlighting the difficulty in travel between major population centers in central Africa. In contrast, the strong interconnectivity between population centers across the east African region since colonial times has likely fostered the rapid growth of the epidemic in this locale.

Conclusion

This study illustrates how phylodynamic analysis of pathogens informed by geospatial data can provide a more holistic and evidence-based interpretation of past epidemics. We advocate that this ‘landscape phylodynamics’ approach has the potential to provide a framework both to understand epidemics' spread and to design optimal intervention strategies.

Little is known about the HIV-1 epidemic in Balkan countries. To fill the gap, we investigated the viral genetic diversity in Bulgaria, by sequencing and phylogenetic characterization of 86 plasma samples collected between 2002 and 2006 from seropositive individuals diagnosed within 1986–2006. Analysis of pol gene sequences assigned 51% of the samples to HIV-1 subtype B and 27% to subtype A1. HIV-1 subtype C, F, G, H, and a few putative recombinant forms were also found. Phylogenetic and molecular clock analysis showed a continuous exchange of subtype A and B between Bulgaria and Western as well as other Eastern European countries. At least three separate introductions of HIV-1 subtype A and four of HIV-1 subtype B have occurred within the past 25 years in Bulgaria. The central geographic location of Bulgaria, the substantial genetic heterogeneity of the epidemic with multiple subtypes, and the significant viral flow observed to and from the Balkan countries have the potential to modify the current HIV-1 epidemiological structure in Europe and highlight the importance of more extensive and continuous monitoring of the epidemic in the Balkans.

Crystals of the human immunodeficiency virus 1 subtype C protease complexed with indinavir and nelfinavir have been grown in the monoclinic space group P21 and shown to diffract X-rays to 2.3 Å resolution.

Crystals of the human immunodeficiency virus 1 (HIV-1) subtype C protease (PR) complexed with the clinically used inhibitors indinavir (IDV) and nelfinavir (NFV) have been grown in the monoclinic space group P21, with mean unit-cell parameters a = 46.7 (±0.1), b = 59.8 (±0.3), c = 87.0 (±0.4) Å, β = 95.2 (±0.5)°. The crystals of both complexes have been shown to diffract X-rays to 2.3 Å resolution. The diffraction data for the subtype C PR complexes with IDV and NFV were subsequently processed and reduced, with overall R
sym values of 8.4 and 11.4%, respectively. Based on the unit-cell volumes, molecular-replacement results and packing considerations, there are two protease homodimers per crystallographic asymmetric unit in each of the complexes. The data were initially phased using a model based on the crystal structure of HIV-1 subtype B PR; the structures have been determined and further refinement and analysis are in progress. These structures and subsequent studies with other inhibitors will greatly aid in correlating the amino-acid variation between the different HIV PRs and understanding their differential sensitivity and resistance to current drug therapy.

Macrophages provide an interface between innate and adaptive immunity and are important long-lived reservoirs for Human Immunodeficiency Virus Type-1 (HIV-1). Multiple genetic networks involved in regulating signal transduction cascades and immune responses in macrophages are coordinately modulated by HIV-1 infection.

Methodology/Principal Findings

To evaluate complex interrelated processes and to assemble an integrated view of activated signaling networks, a systems biology strategy was applied to genomic and proteomic responses by primary human macrophages over the course of HIV-1 infection. Macrophage responses, including cell cycle, calcium, apoptosis, mitogen-activated protein kinases (MAPK), and cytokines/chemokines, to HIV-1 were temporally regulated, in the absence of cell proliferation. In contrast, Toll-like receptor (TLR) pathways remained unaltered by HIV-1, although TLRs 3, 4, 7, and 8 were expressed and responded to ligand stimulation in macrophages. HIV-1 failed to activate phosphorylation of IRAK-1 or IRF-3, modulate intracellular protein levels of Mx1, an interferon-stimulated gene, or stimulate secretion of TNF, IL-1β, or IL-6. Activation of pathways other than TLR was inadequate to stimulate, via cross-talk mechanisms through molecular hubs, the production of proinflammatory cytokines typical of a TLR response. HIV-1 sensitized macrophage responses to TLR ligands, and the magnitude of viral priming was related to virus replication.

Conclusions/Significance

HIV-1 induced a primed, proinflammatory state, M1HIV, which increased the responsiveness of macrophages to TLR ligands. HIV-1 might passively evade pattern recognition, actively inhibit or suppress recognition and signaling, or require dynamic interactions between macrophages and other cells, such as lymphocytes or endothelial cells. HIV-1 evasion of TLR recognition and simultaneous priming of macrophages may represent a strategy for viral survival, contribute to immune pathogenesis, and provide important targets for therapeutic approaches.

A series of HIV-1 protease mutants have been designed to analyze the contribution to drug resistance provided by natural polymorphisms as well as therapy-selective (active and non-active site) mutations in the HIV-1 CRF_01 A/E (AE) protease when compared to the subtype-B (B) protease. Kinetic analysis of these variants using chromogenic substrates showed differences in substrate specificity between pre-therapy B and AE proteases. Inhibition analysis with ritonavir, indinavir, nelfinavir, amprenavir, saquinavir, lopinavir, and atazanavir revealed that the natural polymorphisms found in A/E can influence inhibitor resistance. It was also apparent that a high level of resistance in the A/E protease, as with B protease, is due to aquiring a combination of active site and non-active site mutations. Structural analysis of atazanavir bound to a pre-therapy B protease showed that the ability of atazanavir to maintain its binding affinity to variants containing some resistance mutations is due to its unique interactions with flap residues. This structure also explains why the I50L and I84V mutations are important in decreasing the binding affinity of atazanavir.

HIV-1 epidemic in Western Europe is largely due to subtype B. Little is known about the HIV-1 in Eastern Europe, but a few studies have shown that non-B subtypes are quite common. In Albania, where a recent study estimated a ten-fold increase of AIDS incidence during the last six years, subtype A and B account for 90% of the know infections.

Methodology/Principal Findings

We investigated the demographic history of HIV-1 subtype A and B in Albania by using a statistical framework based on coalescent theory and phylogeography. High-resolution phylogenetic and molecular clock analysis showed a limited introduction to the Balkan country of subtype A during the late 1980s followed by an epidemic outburst in the early 1990s. In contrast, subtype B was apparently introduced multiple times between the mid-1970s and mid-1980s. Both subtypes are growing exponentially, although the HIV-1A epidemic displays a faster growth rate, and a significantly higher basic reproductive number R0. HIV-1A gene flow occurs primarily from the capital Tirane, in the center of the country, to the periphery, while HIV-1B flow is characterized by a balanced exchange between center and periphery. Finally, we calculated that the actual number of infections in Albania is at least two orders of magnitude higher than previously thought.

Conclusions/Significance

Our analysis demonstrates the power of recently developed computational tools to investigate molecular epidemiology of pathogens, and emphasize the complex factors involved in the establishment of HIV-1 epidemics. We suggest that a significant correlation exists between HIV-1 exponential spread and the socio-political changes occurred during the Balkan wars. The fast growth of a relatively new non-B epidemic in the Balkans may have significant consequences for the evolution of HIV-1 epidemiology in neighboring countries in Eastern and Western Europe.

During HIV-1 infection coreceptor switch from CCR5- (R5)- to CXCR4 (X4)-using viruses is associated with disease progression. X4 strains of HIV-1 are highly cytopathic to immature thymocytes. Virtually no studies have evaluated the HIV-1 quasispecies present in vivo within thymic and lymphoid tissues or the evolutionary relationship between R5 and X4 viruses in tissues and peripheral blood.

Methodology/Principal Findings

High-resolution phylodynamic analysis was applied to virus envelope quasispecies in longitudinal peripheral blood mononuclear cells (PBMCs) and lymphoid and non-lymphoid tissues collected post mortem from therapy naïve children with AIDS. There were three major findings. First, continued evolution of R5 viruses in PBMCs, spleen and lymph nodes involved multiple bottlenecks, independent of coreceptor switch, resulting in fitter quasispecies driven by positive selection. Second, evolution of X4 strains appeared to be a sequential process requiring the initial fixation of positively selected mutations in V1-V2 and C2 domains of R5 variants before the emergence of high charge V3 X4 variants. Third, R5 viruses persisted after the emergence of CXCR4-using strains, which were found predominantly but not exclusively in the thymus.

Conclusions/Significance

Our data indicate that the evolution of X4 strains is a multi-step, temporally structured process and that the thymus may play an important role in the evolution/amplification of coreceptor variants. Development of new therapeutic protocols targeting virus in the thymus could be important to control HIV-1 infection prior to advanced disease.

Host cell range, or tropism, combined with coreceptor usage defines viral phenotypes as macrophage tropic using CCR5 (M-R5), T-cell-line tropic using CXCR4 (T-X4), or dually lymphocyte and macrophage tropic using CXCR4 alone or in combination with CCR5 (D-X4 or D-R5X4). Although envelope gp120 V3 is necessary and sufficient for M-R5 and T-X4 phenotypes, the clarity of V3 as a dominant phenotypic determinant diminishes in the case of dualtropic viruses. We evaluated D-X4 phenotype, pathogenesis, and emergence of D-X4 viruses in vivo and mapped genetic determinants in gp120 that mediate use of CXCR4 on macrophages ex vivo. Viral quasispecies with D-X4 phenotypes were associated significantly with advanced CD4+-T-cell attrition and commingled with M-R5 or T-X4 viruses in postmortem thymic tissue and peripheral blood. A D-X4 phenotype required complex discontinuous genetic determinants in gp120, including charged and uncharged amino acids in V3, the V5 hypervariable domain, and novel V1/V2 regions distinct from prototypic M-R5 or T-X4 viruses. The D-X4 phenotype was associated with efficient use of CXCR4 and CD4 for fusion and entry but unrelated to levels of virion-associated gp120, indicating that gp120 conformation contributes to cell-specific tropism. The D-X4 phenotype describes a complex and heterogeneous class of envelopes that accumulate multiple amino acid changes along an evolutionary continuum. Unique gp120 determinants required for the use of CXCR4 on macrophages, in contrast to cells of lymphocytic lineage, can provide targets for development of novel strategies to block emergence of X4 quasispecies of human immunodeficiency virus type 1.